1. Field of the Invention
The present invention relates to a method of producing a three-dimensional tissue in which cell layers are laminated and also to a method of producing an extracellular matrix.
The present disclosure relates to subject matter contained in priority Japanese Application No. 2006-56836, filed on Mar. 2, 2006, which is herein expressly incorporated by reference in its entirety.
2. Description of Related Art
In recent years, regenerative medicine has been attracting great attention. Regenerative medicine has become feasible and actually is in the stage of clinical application with regard to regeneration of tissues with a relatively simple structure, such as bone, cartilage, and skin. However, organs such as kidney and liver have a complex three-dimensional tissue structure composed of many types of cells, and hence, unlike the case of skin or the like, it is difficult to construct the tissues of such organs merely by cell culture. Accordingly, regenerative medicine with regard to regeneration of tissues of such organs still is in the stage of fundamental research. In particular, when cells are cultured artificially, they proliferate two-dimensionally (i.e., in the plane direction) but hardly proliferate three-dimensionally (i.e., in the height direction), which makes it difficult to realize three-dimensional tissue construction with cells merely by cell culture.
Thus, at present, as a method of constructing a tissue with cells, methods of achieving three-dimensional tissue construction by laminating cell sheets each composed of cells having proliferated two-dimensionally have been reported, for example (see JP 2004-261532 A, JP 2004-261533 A, and JP 2005-608 A, for example). For example, according to the method disclosed in JP 2004-261532 A, cells are first cultured on a support coated with a temperature-responsive polymer to induce two-dimensional cell proliferation, thereby forming a single cell layer. Subsequently, the thus-formed cell layer is adhered to a carrier and then the cell layer is separated from the support together with the carrier. Thus, a cell sheet is provided. By laminating a plurality of the thus-obtained cell sheets, three-dimensional tissue construction with the cells is achieved. Furthermore, according to the method disclosed in JP 2004-261533 A or JP 2005-608 A, a fibrin gel layer is formed on a support, and two-dimensional cell proliferation is induced on the fibrin gel layer. Thereafter, the laminate of the fibrin gel layer and the cell layer is separated from the support, thus providing a cell sheet. By laminating a plurality of the thus-obtained cell sheets, three-dimensional tissue construction with the cells is achieved.
However, since the cell layer itself has a very low mechanical strength, it is difficult to separate the cell layer from the support together with the carrier or the fibrin gel layer without losing the shape of the cell layer resulting from the culture on the support. Due to this, in the latter method, the fibrin gel layer with a sufficient mechanical strength is used to facilitate the separation of the fibrin gel layer on which the cell layer is formed. However, in the case where the mechanical strength of the fibrin gel layer is improved by increasing its thickness, there arises a problem in that the thick fibrin gel layer intervenes between the cell layers. When the thick fibrin gel layer intervenes between the cell layers as described above, a time lag or variation may be caused in signal transduction between the respective cell layers because the passage of liquid factors is affected by the diffusion in the gel, for example. Moreover, the intervention of the thick fibrin gel layer may bring about the risk that, for example, after the laminate of the cell sheets has been grafted in a living body, the vascular invasion, which is very important in supplying nutrients and enzymes to the cells inside the laminate, is inhibited by the fibrin gel layer, which may lead to necrosis of the cell layers included in the laminate.
Furthermore, the methods that require separating the cell sheet or laminating the plurality of cell sheets as described above also have a problem in that construction of tissues having a complex shape is difficult. That is, when a tissue to be constructed has a complex shape, the planar shape of a cell sheet to be used also becomes complex. However, separating the cell sheet with high reproducibility while maintaining such a complex planar shape itself is very difficult. Laminating the cell sheets first and then cutting the resultant laminate into a desired shape also can be considered. This, however, still poses a problem because there is a limit on the shape that can be achieve by cutting. In the case where a tissue to be constructed specifically is a tissue with a hollow shape, such as a blood vessel, for example, one may consider laminating the cell sheets and then forming the resultant laminate into a tubular shape by rolling the laminate and adhering its end portions with some means, for example. However, such a technique is very complicated and thus is not practical. Other than the above, various attempts have been made on the construction of three-dimensional tissues. However, there have been problems in that, for example, tissue construction using different types of cells, tissue construction into a desired size, etc. are difficult (see JP 2005-278608 A; Yamada N, Okano Y, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y, Macromol Chem Rapid Commun. 11, 571-576, 1990; Takei, R., Suzuki, D., Hoshiba, T., Nagaoka, M., Seo, S. J., Cho, C. S., Akaike, T., Role of E-cadherin Molecules in Spheroid Formation of Hepatocytes Adhered on Galactose-Carrying Polymer as an Artificial Asialoglycoprotein Model. Biotechnology Letters (2005), 27 (16), 1149-1156; and J. M. Jessup 1, T. J. Goodwin 2, G. Spaulding, Prospects for use of microgravity-substrate d bioreactors to study three-dimensional host-tumor interactions in human neoplasia. Journal of Cellular Biochemistry (1993), 51, 290-300, for example).